Dirk Werling

Identification and functional characterization of a bovine orthologue to DC‐SIGN

Dendritic cell‐specific ICAM‐3‐grabbing nonintegrin (DC‐SIGN) C‐type lectin is almost exclusively expressed at the cell surface of DC. In addition to its normal function facilitating contact of DC with T cells, DC‐SIGN has been shown to bind a variety of pathogens, including Mycobacterium bovis, and HIV‐1 envelope protein gp120. In this study, we identified the bovine ortholog of the human DC‐SIGN gene within the bovine genome, which exists as a single copy. PCR amplified a product, showing a 100% match with the predicted sequences as well as a sequence predicted to be similar to that of SIGNR7. Furthermore, a protein with the same molecular weight as human DC‐SIGN was detected by Western blot in cell lysate derived from bovine DC. To characterize this molecule functionally, the uptake of FITC‐labeled OVA and FITC‐labeled gp120 (FITC‐gp120) by bovine and human DC was assessed. FITC‐gp120 was shown to bind to bovine DC in a time‐ and temperature‐dependent manner. Binding was blocked by a polyclonal anti‐DC‐SIGN antibody but not by a control antibody. Furthermore, blocking of this molecule also reduced the binding of M. bovis bacillus Calmette‐Guerin expressing GFP. Confocal microscopy showed that DC‐SIGN was expressed on the surface of bovine DC. Subsequent pulse‐chase studies revealed that FITC‐gp120 was internalized by bovine monocyte‐derived DC as early as 10 min. Thus, there is evidence of a DC‐SIGN‐like molecule expressed specifically by bovine DC. This molecule may play an important role in the infection of bovine (DC) cells with M. bovis.

Identification of a lineage negative cell population in bovine peripheral blood with the ability to mount a strong type I interferon response.

Lineage negative dendritic cells, or natural interferon-producing cells (NIPC), also referred to as plasmacytoid dendritic cells (pDC) constitute a small population of leukocytes secreting high levels of type I interferon (IFNα/β) in response to certain danger signals. Here, we provide initial data towards the identification of so far uncharacterised circulating bovine pDC like cells. A lineage negative cell population (LIN(-) cells) was isolated from PBMC which showed characteristics similar to that of pDC in other species. Isolated LIN(-) cells presented lymphoid morphology with a semi-crescent nucleus, extensive ER and Golgi network; indicative of pDC. In addition phenotypic analysis of LIN(-) cells described them as distinct from other bovine DC subsets; expressing both lymphoid and myeloid surface markers. LIN(-) cells did not express lineage specific markers, but were MHC class II(+), CD45RO(+), CD80/86(+), CD6(+), WC1(+), CD26(+) and expressed the myeloid markers CD205, CD172a and CD11a. In keeping with pDC, LIN(-) cells express TLR7 mRNA transcripts; however, in a resting state do not express TLR8 or TLR9. Functionally, LIN(-) cells, but not PBMC, monocytes and monocyte derived DC produce large amounts of IFNα/β in response to different CpG oligonucleotides. Taken together, we present data suggesting that an enriched circulating population of bovine LIN(-) cells are uniquely capable of producing IFNα/β in response to CpG oligonucleotides and thus this population likely contain the functional equivalent of bovine pDC.

Replacement of two aminoacids in the bovine Toll-like receptor 5 TIR domain with their human counterparts partially restores functional response to flagellin

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.

Differential Macrophage Function in Brown Swiss and Holstein Friesian Cattle

There is strong evidence that high yielding dairy cows are extremely susceptible to infectious diseases, and that this has severe economic consequences for the dairy industry and welfare implications. Here we present preliminary functional evidence showing that the innate immune response differs between cow breeds. The ability of macrophages (MØ) to kill pathogens depends in part on oxygen-dependent and independent mechanisms. The oxygen-dependent mechanisms rely on the generation of reactive oxygen and nitrogen species (ROS/RNS, respectively). ROS production has been shown to activate the inflammasome complex in MØ leading to increased production of the pro-inflammatory cytokine Interleukin-1β (IL-1β). Conversely RNS inhibits inflammasome mediated IL-1β activation, indicating a division between inflammasome activation and RNS production. In the present study MØ from Brown Swiss (BS) cattle produce significantly more RNS and less IL-1β when compared to cells from Holstein Friesian (HF) cattle in response to bacterial or fungal stimuli. Furthermore, BS MØ killed ingested Salmonella typhimurium more efficiently, supporting anecdotal evidence of increased disease resistance of the breed. Inhibition of autophagy by 3-methyladenine (3-MA) stimulated IL-1β secretion in cells from both breeds, but was more pronounced in HF MØ. Blocking RNS production by l-arginase completely abolished RNS production but increased IL-1β secretion in BS MØ. Collectively these preliminary data suggest that the dichotomy of inflammasome activation and RNS production exists in cattle and differs between these two breeds. As pattern recognition receptors and signaling pathways are involved in the assessed functional differences presented herein, our data potentially aid the identification of in vitro predictors of appropriate innate immune response. Finally, these predictors may assist in the discovery of candidate genes conferring increased disease resistance for future use in combination with known production traits.

Effect of the synthetic Toll-like receptor ligands LPS, Pam3CSK4, HKLM and FSL-1 in the function of bovine polymorphonuclear neutrophils.

Toll-like receptors (TLR) are a family of pattern recognition receptors that sense microbial associated molecular patterns (MAMP) such as microbial membrane components and nucleic acids of bacterial origin. Polymorphonuclear neutrophils (PMN) are the first cell of the innate immune system to arrive at the site of infection or injury and elicit oxidative and non-oxidative microbicidal mechanisms. Observations in human and mouse suggest that TLR ligands can induce direct responses in PMN. So far, there is no information of the effect of synthetic TLR ligands on the response of bovine PMN. The objective of this study was to evaluate the functional response of bovine PMN incubated with four synthetic TLR ligands: ultrapure LPS (TLR4), Pam(3)CSK(4) (TLR2/1), HKLM (TLR2) and FSL-1 (TLR2/6). The results show that all the ligands increment cells size as identified by changes in the FSC-SSC as part of the flow cytometric analysis. Interestingly, only Pam(3)CSK(4) consistently induced a calcium influx, increased ROS production and secretion of gelatinase granules, whereas no response was seen using other ligands. Furthermore, exposure of bovine PMN to ultrapure LPS, Pam(3)CSK(4), HKLM or FSL-1 for 24 hours did not impact on apoptosis of these cells. Our data provide evidence for a selective response of bovine PMNs to TLR ligands.

Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response

Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

Bovine Viral Diarrhea Virus Strain- and Cell Type-Specific Inhibition of Type I Interferon Pathways

The interaction of noncytopathogenic bovine viral diarrhea virus type 1 (BVDV-1ncp) with antigen-presenting cell (APC) subsets is of great interest due to the recent increase in severe acute BVDV outbreaks attributed to this genotype (1, 12, 13). We report the effect of a BVDV-1ncp strain (Ho916ncp [13]) causing severe acute infection on in vitro type I interferon (alpha/beta interferon [IFN-α/β]) production compared to that of a mild-acute strain (Ky1203ncp) (15). Ho916ncp but not Ky1203ncp induced IFN-α/β in only lineage-negative (LIN−) cells, a recently described enriched IFN-α/β-producing cell population in cattle (A. Gibson, S. Miah, P. Griebel, J. Brownlie, and D. Werling, submitted for publication). In agreement with published data, monocytes and monocyte-derived dendritic cells (moDC) did not produce IFN-α/β in response to either strain (4, 11, 17). n nExperimental infection of calves with BVDVncp results in the detection of IFN-α/β and the IFN-α/β-inducible gene Mx (5, 20) as well as serum IFN-α/β activity in pregnant dams and the fetus (22). The identification of IFN-α/β-producing cells within the lymph nodes of BVDVncp-infected calves (4) and of enriched circulating IFN-α/β-producing cells (Gibson et al., submitted) points to an APC subset as a cellular source of IFN-α/β during in vivo BVDVncp infections. In the present study, monocytes, moDC, and LIN− cells, generated from the same animal (11, 24-26, 28; Gibson et al., submitted), were exposed to either strain at a multiplicity of infection (MOI) of 0.1 and IFN-α/β was assessed after 48 h, using a previously described Mx-CAT (chloramphenicol acetyltransferase) reporter assay (4-6, 9). LIN− cells, but not monocytes or moDC, produced significant amounts of IFN-α/β in response to Ho916ncp but not to Ky1203ncp or mock-infected control (Fig. u200b(Fig.1).1). To assess whether these observed differences in IFN-α/β production were due to differences in viral replication, the presence of BVDV was determined by immunoperoxidase staining using a bovine hyperimmune serum, similar to what was described previously (11). Astonishingly, no clear differences in abilities to infect the different cell types were seen for the two BVDVncp strains (Fig. u200b(Fig.2).2). As differences in viral replication did not explain differences in IFN-α/β production, we next assessed the potential effects of both BVDVncp strains on interferon response factors (IRFs). BVDVncp is known to inhibit IFN-α/β production through viral proteins Npro (2, 3, 10, 14) and Erns (16, 18, 19), which block IFN-α/β production by targeting IRF-3 for degradation by polyubiquitination (7, 14) or by degrading viral RNA (19). Translocation of IRF-3 occurs during infection but not binding to DNA (2). In contrast, IRF-7 is neither activated nor translocated to the nucleus (2). n n n nFIG. 1. n nType I interferon response to BVDVncp. Monocytes (MON), moDC, and LIN− cells (LIN) were isolated from the same animal and stimulated with 50 μg ml−1 of poly(I:C), BVDVncp strains Ho916ncp and Ky1203ncp at an MOI of 0.1, and corresponding ... n n n n n nFIG. 2. n nAntigen-presenting cell susceptibility to BVDVncp. Monocytes (A), moDC (B), and LIN− cells (C) were infected with BVDVncp strains Ho916ncp and Ky1203ncp for 48 h (MOI, 0.1). Cells were fixed and stained for the presence of BVDV antigen using a ... n n n nWe assessed the impact of both strains on IFN-α/β signaling components IRF-3 and IRF-7 by Western blotting, which showed that IRF-3 and IRF-7 were increased in all three cell types infected with Ho916ncp compared to mock-infected controls. In contrast, Ky1203ncp induced a reduction of IRF-7 expression in monocytes and moDC and a reduction in IRF-3 expression in all three cell types. IRF-7 remained unchanged in LIN− cells, potentially indicating cell type-specific degradation in monocytes and moDC for Ky1203ncp (Fig. u200b(Fig.3).3). Ho916ncp-induced IFN-α/β production by LIN− cells appears to be independent of IRF-3 or IRF-7 modulation; however, LIN− cells, unlike monocytes or moDC, express TLR7 (27; Gibson et al., submitted), which has been recently implicated in the recognition of other pestiviruses, such as West Nile virus and dengue virus (8, 21, 23). Ho916ncp, as a severe acute BVDV-1ncp strain could be more readily accessible to the endosomal compartment within LIN− cells, thus producing cell type-restricted IFN-α/β through TLR7. Our data show for the first time differences in BVDV-1ncp strains with regard to a cell-specific IFN-α/β response and offer some suggestions for this modulation. n n n nFIG. 3. n nRelative expression of IRF-3 and IRF-7 in response to BVDVncp. Monocytes (MON), moDC, and LIN− (LIN) cells were isolated from the same animal and stimulated with Ho916ncp and Ky1203ncp alongside mock-infected controls for 48 h. Protein lysates ...

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Structural characterisation of Toll-like receptor 1 (TLR1) and Toll-like receptor 6 (TLR6) in elephant and harbor seals.

Pinnipeds are a diverse clade of semi-aquatic mammals, which act as key indicators of ecosystem health. Their transition from land to marine environments provides a complex microbial milieu, making them vulnerable to both aquatic and terrestrial pathogens, thereby contributing to pinniped population decline. Indeed, viral pathogens such as influenza A virus and phocine distemper virus (PDV) have been identified as the cause of several of these mass mortality events. Furthermore, bacterial infection with mammalian Brucella sp. and methicillin-resistant Staphylococcus aureus strains have also been observed in marine mammals, posing further risk to both co-habiting endangered species and public health. During these disease outbreaks, mortality rates have varied amongst different pinniped species. Analyses of innate immune receptors at the host-pathogen interface have previously identified variants which may drive these species-specific responses. Through a combination of both sequence- and structure-based methods, this study characterises members of the Toll-like receptor (TLR) 1 superfamily from both harbour and elephant seals, identifying variations which will help us to understand these species-specific innate immune responses, potentially aiding the development of specific vaccine-adjuvants for these species.

Of creatures great and small: the advantages of farm animal models in immunology research.

In recent decades the inappropriate use of antibiotics in farm animals, either as growth enhancers or for treatment of infectious diseases, has resulted both in higher concentrations of antibiotic residues in meat destined for the human food chain and in an increasing prevalence of antibiotic-resistant bacteria (Carlet et al., 2012). Increased public concerns regarding their potential impact on human health may result in a reduction or cessation in the use of antibiotics in farm animals (Veterinary Record, 2011, 2012). Thus, alternative treatments for the control of infectious diseases in farm animals need to be identified as a research priority. In achieving this goal, the physiological relevance of an animal model to the intended target population becomes a key factor. The majority of models currently used for pharmaceutical research are murine and it is possible that potentially valuable therapeutics are discounted on the basis of lack of responses generated in this system, as shown very recently for sepsis (Seok et al., 2013). Several non-rodent species, such as cattle, pigs, and chicken, are both valuable models for infectious diseases in humans (Hein and Griebel, 2003; Visscher and Goddard, 2011; Waters et al., 2011; Costa et al., 2012; Meurens et al., 2012) and important clinical targets in their own right. Effective vaccines have been developed for many acute viral and bacterial infections, whereas for others, like tuberculosis (TB), there is still a need for reliable vaccines. Pathology seen in the murine models of TB currently used is very different to the pathology seen in humans (Gupta and Katoch, 2009), whereas the bovine and human diseases share many similarities (Waters et al., 2011). For such diseases it is crucial that we use natural disease-models, with the appropriate pathogen in the most appropriate host, to examine the host-pathogen interactions that occur in outbred populations. A fundamental point in the development of new and improved intervention strategies is the understanding of host differences in the innate immune response, which primes the subsequent adaptive immune response. Although the innate immune system has been largely conserved during evolution, marked variations and diversity exist between different mammalian species within Pattern-Recognition-Receptor (PRR) structure (Jungi et al., 2011). These differences are based on evolutionary pressure within the innate immune system, potentially reflecting the specific threats encountered by each species (Zhang et al., 2010). This selective pressure appears to be absent in the available murine sequences (Werling et al., 2009). The similarity between human and farm animal PRR (Jungi et al., 2011) is further supported by their similar response to ligands (Kapetanovic et al., 2012), in contrast to murine PRR (Hajjar et al., 2002; Grabiec et al., 2004; Farhat et al., 2010). Since recognition by PRR is associated with adaptive immunity by providing optimal immunostimulation, learning more about these key molecules in farm animals might inform us about their adjuvant effect in vaccines for use in these animals as well as humans. Given the size and blood volumes of farm animals, there are also greater opportunities to repeatedly access to different cell types – an asset which would facilitate the assessment of cell specific effects of such immunomodulatory agents on autologous cells (Hein and Griebel, 2003). Farm animal models do have their disadvantages such as dedicated housing, biosecurity, and the confinement of infected animals. However, it is misleading to rely on murine models to fill the gaps in our knowledge of disease-pathogenesis, and choosing a research model should be more than just a matter of convenience and convention (Bolker, 2012). n nHowever, in addition to their potential use as models for human diseases, research in farm-animals models have a “right-on-their-own,” taken the predicted increase in food supply necessary by 70% to support an ever-increasing global population as well as the occurrence of new emerging diseases, such as Schmallenberg virus into account. These challenges can be faced and overcome by an increased readiness of farm animal research centers to apply their models in novel ways, a willingness of the medical profession to accept more suitable disease-models, and a willingness of funding agencies and companies to invest in this type of research partnership.